Type 1A diabetes (T1D) is a chronic disorder that results
from the immune-mediated destruction of the insulin-producing ß-cells of the
pancreatic islets1. In its initial phase, which is
clinically silent, T lymphocytes and other inflammatory cells invade the islets
and eventually destroy them. The disease then becomes clinically evident with
the pathological consequences (hyperglycemia, ketosis and long-term
complications) resulting from the inability to maintain glucose and lipid
homeostasis.

Type
1 diabetes is a T-cell mediated disease

The first indication that T1D is an autoimmune disease came from the results of
a comprehensive histological examination of pancreata from diabetic patients
who had died shortly after diagnosis. This showed that most of the subjects had
significant lymphocytic infiltration of their islets concordant with loss of
ß-cell mass 2. Subsequently, islet-cell
antibodies (ICAs) and anti-pancreatic cell-mediated immunity were detected in
recently diagnosed T1D patients 3-5, suggesting that the lymphocytes
accumulated as a result of attraction by antigens derived from pancreatic ß-cells6. Consistent with this hypothesis,
insulitis was only seen in islets containing ß-cells. With the advent of
monoclonal antibodies capable of identifying distinct lymphocyte
sub-populations more detailed immunohistochemical examinations of islet
infiltrates became possible. One of the earliest of such studies showed a
predominance of CD8+ T-cells in the islets of a deceased 12-year old
girl with newly diagnosed T1D, which, together with the observed up-regulation
of MHC class I molecules by islet cells, implicated cytotoxic T-cells (CTLs) in
ß-cell destruction 7. Additional studies of pancreas
from patients with type 1 diabetes have confirmed preponderance of CD8 T-cells
and the presence of B-lymphocytes related to extent of ß-cell destruction 8. The JDRF nPOD (Network for
Pancreatic Organ Donors with Diabetes) program now allows viewing of pancreatic
histology of cadaveric donors directly online (http://www.jdrfnpod.org/).
Similarly, the strong association of T1D with particular MHC II haplotypes (see
chapter 7) suggested a critical role for CD4+ T-cells in the disease
process (reviewed by 9. Additional circumstantial evidence
supporting a crucial role for T-cells and MHC-restricted self antigen
recognition in diabetogenesis came from the reversal and recurrence of diabetes
following twin to twin pancreatic isografts 10,
11, and the inadvertent transfer of
disease between HLA-identical siblings by bone marrow transplantation 12.

The mere presence of T-cells in infiltrates, though highly
suggestive, does not by itself establish a direct role for these cells in the
development of T1D. However, the histological findings were subsequently
followed by reports of T-cell reactivity to ß-cell proteins, providing further
support for the hypothesis. Thus, Roep and colleagues established CD4+ T-cell
lines and clones restricted to HLA-DR from the peripheral blood of new-onset
diabetics after stimulation in vitro
with rat insulinoma cells 13.Of the eight clones examined, five appeared to recognize insulinoma
membrane components, one of which was a 38kD protein later termed IMOGEN38 14-18. Surprisingly, after expression
cloning IMOGEN38 was shown to be a broadly distributed mitochondrial protein (a
probable subunit of the mitochondrial ribosome), and studies with the human
ortholog suggested that the response was likely xenogeneic (JC Hutton personal
communication). Nonetheless, the identification of several islet cell molecular
targets allowed subsequent studies to be conducted using defined autoantigens,
rather than crude fractions, and have suggested that the peripheral blood of
diabetic subjects and their at-risk relatives contain elevated numbers of
T-cells able to recognize epitopes from ß-cell proteins 19. Such studies suggest that T-cells
provide a legitimate therapeutic target for intervention, and results from clinical
trials of new-onset diabetic patients withhumanized anti-CD3 monoclonal antibodies delayed the deterioration of
circulating C-peptide levels normally seen in the year following diagnosis in 9
of 12 subjects 20-22. Attempts to build upon these
partial successes and improve the therapeutic regimen are currently in
progress.

Animal models of T1D

A. Spontaneous models

Since the target organs of T1D (islets and draining pancreatic lymph nodes) are
inaccessible in human subjects, the study of T1D has been greatly facilitated
by the availability of animal models such as the Biobreeding-diabetes prone
(BB-DP) rat 23 and the nonobese diabetic (NOD)
mouse 24, which spontaneously develop
diseases that mimic many features of human T1D. In particular, the NOD mouse has
been the subject of extensive studies for over 20 years 25-27, and has provided key experimental
evidence supporting several strategies to treat the human disease, some of
which are showing promise in initial clinical trials 28,
29. Numerous abnormalities have been
reported in the immune systems of NOD mice, including defects in antigen
presenting cells (APCs) 30 and hyporesponsiveness of T
lymphocytes 31,
32, which together may compromise both
central 33and peripheral 34 tolerance to pancreatic ß-cells.
Similar abnormalities have been reported in human subjects (for example 35-37, and it has been proposed that
T-cell hyporesponsiveness may be a general feature conferring susceptibility to
inflammatory autoimmune disorders 38. However, it must be noted that the
immune systems of humans and mice show several key differences (reviewed by 39, which must be kept in mind when
extrapolating between these species 28,
40-42. Indeed, there are notable
differences between T1D in NOD mice and humans, not the least being that NOD
mice only develop disease if kept in specific pathogen free conditions.
Moreover, in these animals disease is associated with pronounced cellular
infiltrates that surround the individual islets (Figure 1) that begin to form
at least 7 weeks prior the onset of overt

disease 43,
44. In contrast, pancreata obtained at
post mortem from diabetic subjects who died shortly after clinical
manifestation of T1D typically show a much less florid infiltration (e.g. 7,
45). In that NOD mice are inbred they
can only be considered to be the equivalent of a single genotype "case
study" of T1D and a genotype that is homozygous at all loci.

Although
the precise sequence of events that lead to T1D in NOD mice remain uncertain,
the central role of T-cells in diabetogenesis in these animals is
incontrovertible. Thus, treatment of newly diabetic animals with anti-CD3
antibodies, which suppress immune responses by transient T-cell depletion and
modulation of T-cell Receptor (TCR) signaling, induces long-term remission 46,
47.Moreover, diabetes can be transferred to immuno-compromised hosts by
mixed T-cell populations, or in some instances, individual T-cell clones 48-51. For example, Haskins
and colleagues isolated 8 CD4+ T-cell clones recognizing islet
antigens (including chromagranin and islet amyloid polypeptide) 52,
53 presented by the NOD MHC class II
molecule, I-Ag7, at least 5 of which are capable of inducing disease
after adoptive transfer 54,
55. Transgenic mice expressing the
T-cell receptor (TCR) of one of the diabetogenic clones, BDC2.5 (target peptide
derived from chromogranin), have been generated and bred onto the NOD
background 56. Interestingly, evaluation of the
lymphoid compartment in NOD/BDC2.5 animals showed no sign of negative selection
in the thymus, with normal peripheral T-cell reactivity. This was also true of
transgenic C57BL/6-H-2g7 (B6g7/BDC2.5) animals. However,
autoimmune pathogenesis was highly dependent upon the genetic background of the
animal 57. In both NOD and B6g7
transgenics there was no manifestation of disease in the first 2 weeks of life,
with pancreatic islets completely free of infiltration. Insulitis appeared
abruptly at 18 days, and subsequently progressed to eventually involve
essentially all islets. However, whilst the B6g7/BDC2.5 animals
suffered a highly aggressive insulitis and the majority rapidly progressed to
overt disease, paradoxically, NOD/BDC2.5 animals were significantly protected
from spontaneous disease, and exhibited a more benign "respectful"
insulitis.Nevertheless, young
NOD/BDC2.5 are significantly more sensitive to cyclophosphamide induced
diabetes than their non-transgenic relatives 58, and NOD/scid/BDC2.5 mice rapidly progress to overt T1D and typically die of
diabetic complications on or before 33 days of age 59. The precise mechanisms by which
the un-manipulated NOD/BDC2.5 animals restrain their insulitis are currently
uncertain, but may involve populations of T-cells expressing alternative a T cell
receptor chains due to incomplete allelic exclusion at this locus 56,
60.

In
addition to TCR transgenic animals, targeted gene disruption and retrogenic 61 technologies have also been used to
study other features of T1D in NOD mice. For example, mice lacking expression
of ß2-microglobulin (ß2M), that consequently do not
express functional MHC class I molecules, do not develop insulitis 62-65, implicating CD8+
T-cells in the initiation of disease. Interestingly, transgenic restoration of
MHC class I expression in NOD-ß2M-/- mice using different
ß2M alleles provided contrasting results. Thus, animals
reconstituted with the endogenous ß2Ma allele developed
T1D, whilst those given the ß2Mb allele (which only
differs at a single amino acid residue) did not 66. At present the precise mechanism
of protection is uncertain, although it may reflect subtle differences in the
peptide-binding properties of the resultant MHC class I molecules 67.

Targeted
gene disruption has also provided evidence for a key role for proinsulin in
diabetogenesis in NOD mice. In contrast to humans, mice have 2 non-allelic
insulin genes (insulin 1 on chromosome 19, and insulin 2 on chromosome 7), and
recently NOD mice with disruptions in either one have been created 68,
69. Surprisingly, the mice show
contrasting phenotypes; insulin 1-/- mice are markedly
protected from diabetes (but do develop anti-insulin autoantibodies), while
insulin 2-/- animals show accelerated disease. In contrast, animals
whose sole preproinsulin is a mutant form of insulin 2 in which the
immunodominant B:9-23 epitope is disrupted are completely protected from
diabetes 70, although they do develop sialitis
confirming the organ-specificity of the effect. Such results are at variance
with those from related studies in which NOD mice lacking the autoantigens
GAD65, IA-2, phogrin (IA-2ß) and IGRP 71 were produced and where disease
occurrence was not significantly altered 72-74. This suggests a central role for
(pro) insulin in the disease process, although the precise mechanisms of
acceleration, or protection from disease, remain to be determined. The
potential central role of insulin and insulin peptide B:9-23 is supported by
studies where both insulin genes are eliminated and an insulin mutated at
position B16 (Y to A). These mice do not develop diabetes 70,
75. Krishnamurthy and coworkers have
found that eliminating response to proinsulin eliminates the prominent CD8
T-cell response to IGRP 76,
77. Vignali and coworkers have
introduced a new methodology to study T-cell receptor targeting of islet ß
cells, namely the creation of retrogenic mice 78,
79. The creation of retrogenic mice
utilizes retroviruses to introduce T-cell receptors into bone marrow cells that
are then transplanted into immunodeficient mice. This technology greatly
accelerates studies of such T-cell receptors compared to creating transgenic
mice. Within 8 weeks, the pathogenicity of T-cell receptors can be assessed.
Studying a series of 17 retrogenes, those targeting GAD failed to induce
diabetes. Insulin peptide B:9-23 reactive TCR caused delayed diabetes and
IA-2/phogrin TCR caused insulitis and TCRs targeting chromogranin (e.g. BDC
2.5) caused diabetes as did TCR’s targeting unknown molecules. (Diabetes
induction TCR: BDC-10.1 > BDC-2.5 > NY 4.1 > BDC 6.9).Of note, a series of TCRs targeting GAD65
caused fatal encephalitis independent of the induction of anti-GAD
autoantibodies61.No insulitis was observed.Presumably this was related to the lack of GAD in mouse islets.

B. Experimental autoimmune diabetes
(EAD)

As
only a limited number of animal models exhibiting spontaneous disease are
currently available, systems to induce EAD in non-autoimmune prone mice have
also been developed. Typically these are based upon the transgenic ß-cell
expression of heterologous proteins under control of the rat insulin promotor
(RIP). Such models have provided key insights into the establishment and breaking
of tolerance to ß-cell antigens. Initial studies showed that some lines of
RIP-Tag C57BL/6 mice, which express the SV40 large T antigen (Tag) in their
ß-cells, were intolerant of the transgene, developing spontaneous autoimmunity 80. Tolerance, or autoimmunity,
correlated with the presence or absence of embryonic expression of the
transgene, with animals that did not express Tag until adulthood developing
disease. Moreover, tolerant animals also expressed the transgene in the thymus 81, suggesting that, at least for some
proteins, central tolerance can be established to apparently organ-specific
antigens (reviewed in 82. However, central tolerance could
be broken if the precursor frequency of peripheral autoreactive T-cells was too
high. Thus, tolerant RIP-Tag mice crossed with transgenic mice showing low
expression (~10% of peripheral T-cells) of the TCR of a Tag specific CTL were
protected from spontaneous disease 83. In contrast, offspring of parents
showing high expression (~90%) of the transgenic TCR were intolerant.The importance of central tolerance was also
demonstrated in a model where CBA (H-2k) mice expressing the Kb
MHC class I molecule under control of the RIP were crossed with transgenic mice
expressing the TCR of an H-2k-restricted CTL clone recognizing Kb.
In this case the progeny rejected skin grafts from H-2b animals, but
were protected from T1D. However, crossing the double transgenic mice with
RIP-IL-2 animals caused rapid onset disease 84. Neonatal replacement of the
thymuses of the double transgenic animals with tissue from non-transgenic mice
permitted high avidity Kb-specific T-cells to reach the periphery,
which allowed disease to be triggered in animals primed either with allogeneic
skin grafts or the injection of irradiated splenocytes from H-2b
donors 85. Some animals required multiple
primings, suggesting that the duration of the stimulus, as well as the avidity
of the peripheral autoreactive T-cells, profoundly influences the course of
disease.

The
lack of spontaneous disease in RIP-Kb x Kb-TCR mice after
thymus replacement, despite the presence of peripheral high affinity Kb-specific
T-cells, indicates that protective mechanisms exist in the periphery which can
act to negate a lack of central tolerance. Two potential mechanisms have been
proposed, namely immune ignorance and active tolerance 86, and evidence that both are
involved in protection from T1D has come from RIP-based transgenic models. For
example, two strains of mice secreting either high (RIP-Ovahi) or
low (RIP-Ovalo) levels of ovalbumin from their ß-cells were exposed
to 5, 6-carboxy-succinimidyl-fluorescein-ester (CSFE) labeled OT-1 T-cells
specific for ovalbumin. Analysis of the pancreatic draining lymph nodes 3 days
after transfer revealed that the OT-1 cells had proliferated in the RIP-Ovahi
but not the RIP-Ovalo mice 87, indicating that the level of
antigen acquired by antigen presenting cells (APCs) in the RIP-Ovalo
animals was insufficient to trigger naive T-cells, and consequently that the immune
system was ignorant of the presence of the neo-antigen. Both strains developed
T1D if exposed to pre-activated OT-1 CTLs, confirming that the RIP-Ovalo
animals did indeed exhibit ß-cell expression of the transgene. However, despite
proliferation in the draining lymph nodes, adoptive transfer of even 107
naive OT-1 cells to RIP-Ovahi mice did not induce disease 88, suggesting that effector CTL
generation was inefficient under these conditions. Nonetheless, tolerance to
ß-cell expression of ovalbumin could be broken if the efficiency of
presentation was enhanced. Thus, studies with RIP-mOva mice, which express a
membrane-anchored chimeric fusion protein derived from ovalbumin and the
transferrin receptor 89, demonstrated that cell-associated
ovalbumin was cross-presented to CD8+ T-cells ~50,000-fold more
efficiently than the secreted form 90and adoptive transfer of 5 x 106
naiveOT-1 T-cells into non-irradiated
RIP-mOva mice rapidly induced disease.

To
analyze the fate of OT-1 cells following transfer into RIP-mOva mice without
inducing disease these animals were crossed with bm1 mice (that have a mutation
in the Kb molecule which prevents presentation to OT-1 T-cells 91. The resulting RIP-mOva.bm1 mice
were lethally irradiated and reconstituted with bone marrow from wild-type
C57BL/6 animals so that OT-1 cells could interact with hematopoietic, but not
peripheral, cells. Analysis of draining lymph nodes 3 days after adoptive
transfer of CSFE-labeled OT-1 T-cells confirmed that they had proliferated, but
after 8 weeks transgenic T-cells comprised only ~1% of CD8+ T-cells
recovered from the spleens and lymph nodes of B6®RIP-mOva.bm1 mice as compared to
~7.4% in B6®bm1 animals, suggesting that activation had led to
peripheral deletion